μ slide 8 well ibitreat chambers

Two
irradiation methods were investigated: (i) highly focused NIR spot
(high power densities of a 785 nm laser “pointer” with
diameter ∼5 μm, as in classical optical tweezers)^{44 (link)} for single-cell experiments (exposure times
∼5–10 s) and (ii) illumination of thousands of cells
with a large diameter (∼0.65 cm; low power density) collimated
NIR beam, for which we used a 808 nm laser, for large-area irradiation
aimed to achieve intracellular conversion of substrates into products
(optimal irradiation conditions: 10 W·cm^–2,
1 min). Confocal imaging experiments with living cells were performed
in μ-Slide 8 well-ibiTreat chambers (1 cm²/well,
Ibidi, Germany, no. 80826). Images were captured on an Andor Dragonfly
spinning disk confocal system mounted on a Nikon TiE microscope equipped
with a Zyla 4.2 PLUS camera (Andor, Oxford Instruments) and an OKO-lab
incubator to keep cells at 37 °C during all the experiment. Images
were acquired with different magnification objectives (20×, 60×,
and 100×) and different ex./em. channels (details in the Supporting Information). All the images were
processed with ImageJ.

Flp-In T-REx HeLa cells were grown in μ-Slide 8 Well ibiTreat chambers (ibidi, Martinsried, Germany) in CO₂ independent medium (Thermo Fisher Scientific) with 10% tetracycline-free FBS supplemented with 250 μg ml⁻¹ hygromycin and 4 μg ml⁻¹ blasticidin (Invitrogen). Gene expression was induced by addition of 0.05 μg ml⁻¹ doxycycline (Sigma-Aldrich) for 24 h. Live imaging of cells was done with spinning disk confocal microscopy on a 3i Marianas system (Intelligent Imaging Innovations) equipped with Axio Observer Z1 microscope (Zeiss), a CSU-X1 confocal scanner unit (Yokogawa Electric Corporation), Plan-Apochromat × 100/1.4 oil objective (Zeiss), Orca Flash 4.0 sCMOS Camera (Hamamatsu) and controlled by Slidebook Software 6.0 (Intelligent Imaging Innovations). Image series were acquired as z-sections at 0.27 μm with an interval of ∼1,300 ms for 90 time points and maximal intensity projections were made with Slidebook Software 6.0 (Intelligent Imaging Innovations).